Fig. 3
Establishment of sequential stimulation of cAD-MSCs for small extracellular vesicle (sEV) production. A The schematic diagram demonstrates the protocol for sequential stimulation of cAD-MSCs, as well as the subsequent collection and purification of sEVs. B While CTRL was incubated under normal condition, IVFG was conducted by flowing through with the in vitro fertilization gas (5% O2). ES20 was done by applying a direct current at 20 mV/mm through the platinum electrodes. For IVES, cAD-MSCs underwent IVFG and ES20 consecutively. C Morphological appearances of cAD-MSCs before conditioned medium collection were revealed by a phase-contrast microscope at 200X magnification. The scale bars represent 200 μm. D Viable cells were counted by trypan blue exclusion assay. The fold change in viable cell count was obtained by CTRL normalization. E Expression of mRNA makers for hypoxia activation (Hif1a), calcium homeostasis (Hsp90b1), exosome biogenesis (Syntenin-1) and endolysosomal trafficking (Rab27b) were analyzed via RT-qPCR. The relative mRNA expression was determined by normalization with the reference gene (Gapdh). The fold change in mRNA expression was calculated by normalization with Gapdh and the control condition (CTRL). F Pearson’s correlation matrix of the relative mRNA expressions demonstrates the data distribution as a density plot (diagonal) and the correlation between genes as a pairwise scatter plot (lower diagonal). The linear regression curve was visualized along with a grey region of 95% confidence interval. The Pearson’s correlation coefficient (R) was annotated with statistical significance symbol (*; p-value ≤ 0.05, **; p-value ≤ 0.01). In boxplots, mean values of each group were annotated at the bottom, and global statistics were stated at the top. The bars indicate pairwise comparisons (ns; no significance or p-value > 0.05, *; p-value ≤ 0.05). Abbreviation: CTRL; control condition, IVFG; in vitro fertilization gas pre-conditioning, ES20; electrical stimulation at 20 mV/mm, IVES; sequential stimulation of IVGF and ES20. D1; sEV sample from Day1 collection, D2D3; pooled sEV sample from Day2 and Day3 collections, and AU; arbitrary unit